Polynucleotide comprising sequences of wheat gliadins and use thereof for silencing by RNAi

ABSTRACT

The present invention relates to the specific silencing of the α (alpha), β (beta), γ (gamma) and ω (omega)-gliadins of hard wheat for flour by RNA interference (RNAi) through employment of a polynucleotide which is transcribed into an hpRNA (hairpin RNA). Furthermore the present invention additionally relates to a vector, cell, plant or seed comprising the polynucleotide, the expression whereof is specifically directed in particular tissues of wheat seeds through gene expression-regulating sequences such as, for example, the promoter of a gene of γ-gliadins or the promoter of the gene encoding for a D-hordein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/147,151, filed Nov. 25, 2011, which is a U.S. national phaseapplication filed under 35 U.S.C. § 371 of International Application No.PCT/ES2010/070045, filed Jan. 26, 2010, which claims the benefit of thepriority date of Spanish Application No. P200900302, filed Feb. 3, 2009.The contents of the aforementioned applications are hereby incorporatedby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Dec. 30, 2014, isnamed “SequenceListing.txt” and is 7.27 KB (7,450 bytes) in size.

The present invention concerns the specific silencing of the α (alpha),β (beta), γ (gamma), and ω (omega) gliadins of hard wheat and flour byinterference RNA (iRNA) through use of a polynucleotide that istranscribed into an hpRNA (hairpin RNA). Furthermore, the presentinvention also concerns a vector, cell, plant, or seed comprising thepolynucleotide, the expression whereof is specifically directed inparticular tissues of wheat seeds through gene expression-regulatingsequences such as, for example, the promoter of a gene of γ gliadins orthe promoter of the gene that codes for a D-hordein.

PRIOR ART

RNA interference (RNAi) is a system of degradation of messenger RNAmediated by double-stranded RNA that allows the specific silencing ofparticular genes. Its discovery has made it possible to design vectorscomposed of a promoter and termination signals including the sequence ofthe gene one wishes to silence and having sense and antisense sequencesseparated by a spacer sequence of variable length.

siRNAs (the English abbreviation for small interfering RNA or shortinterfering RNA) are molecules of double-stranded RNA (dsRNA, theEnglish abbreviation for double-stranded RNA) of 21-25 nucleotides (nt)that originate from a longer precursor dsRNA. Precursor dsRNAs may be ofendogenous origin, in which case they are referred to as miRNAs (encodedin the genome of the organism) or of exogenous origin (such as virusesor transgenes). Both siRNA and miRNA are types of iRNA (interferenceRNA). iRNA suppresses the post-transcriptional expression of aparticular mRNA (the English abbreviation for messenger RNA) recognizedby the iRNA sequence.

When a cell receives a dsRNA precursor (single-stranded RNAs do notproduce this effect), which may be generated from an exogenoustransgene, a viral agent, or an endogenous genetic element, it isfragmented into siRNAs through the action of an enzyme referred to asDicer, a cytoplasmic enzyme of the RNAse III family. Dicer cleaves thedsRNA into double-stranded fragments of approximately 21-25 nucleotides(siRNA), with the 5′ end phosphorylated and two unpaired nucleotidesprotruding at the 3′ end. Of the two strands of siRNA, only one,referred to as the guide strand, is incorporated into the enzymaticcomplex RISC (RNA-induced silencing complex), while the other strand isdegraded. The thermodynamic characteristics of the 5′ end of the siRNAdetermine which of the two strands is incorporated into the RISCcomplex. The strand that is less stable at the 5′ end is normallyincorporated as the guide strand, either because it has a higher contentof AU bases or because of imperfect pairings. The guide strand must becomplementary to the mRNA to be silenced in order forpost-transcriptional silencing to occur. Subsequently, the RISC complexbinds to the complementary mRNA of the guide strand of the siRNA presentin the complex, and cleavage of the mRNA occurs. After this, thefragments obtained are degraded. In this manner, the siRNAs causepost-transcriptional silencing of the target nucleotide sequences sothat the protein that would result from expression of these sequences isnot obtained.

The grain proteins in wheat, although contained in lesser amounts(7-18%) than carbohydrates (60-75%), are essential for the functionalproperties of flour. The main grain proteins are the glutenins andgliadins, which make up gluten. The gliadins have been found to beresponsible for the development of celiac disease, as epitopes(antigenic determinants) recognized by the intestinal T cells have beenidentified in regions of a and γ gliadins (Arentz-Hansen, et al., 2002.Gastroenterology 123: 803-809). People suffering from celiac disease areintolerant to gluten. Gluten intolerance is characterized by chronicinflammation of the proximal portion of the small intestine caused byexposure to gliadin. By producing wheat having a sharply reduced gliadincontent, it would be possible to produce a food for celiac diseasepatients.

There are four known types of gliadins: α (alpha), β (beta), γ (gamma),and ω (omega). Suppression of wheat grain gliadins using RNAi technologyhas been used in recent years. To date, it has only been possible tosuppress particular gliadins. For example, by using geneticconstructions that give rise to an RNAi type hairpin having thestructure promoter-sense sequence-spacer-antisense sequence-terminator,it has been possible to eliminate type α gliadins (Folck et al., 2005.XII International Conference on Plant Embryology. Oral presentation) orγ type gliadins (Gil-Humanes et al. 2008. Journal of Cereal Science48(3): 565-568) almost completely, but it has not yet been possible toeffectively eliminate all types of gliadins.

Obtaining plants having seeds with a highly reduced gliadin contentpresents technical difficulties, e.g., first, the increased number ofgenes coding for gliadins, and second, the fact that wheat plants arehexaploid. There are more difficulties involved in achieving stabletransformation in wheat plants than in transformation of any other planthaving fewer copies of the genome.

EXPLANATION OF THE INVENTION

The present invention concerns a polynucleotide comprising two sequencepairs, with each subsequence combining in a particular order to giverise to a sequence whose transcription into RNA is capable of generatinghpRNA (hairpin RNA), e.g., RNA in the shape of a hairpin, adouble-stranded RNA that will be processed by endoribonucleasesdescribed in the prior art, which is used to generate the siRNAs thatcause post-transcriptional silencing of all of the mRNAs (messenger RNA)that code for all types of wheat gliadins. For this purpose, the foursubsequences are: the sense sequence of the ω gliadins, the sensesequence of the α, β, and γ gliadins, and the two previous antisensesequences.

By means of this polynucleotide, whose expression is specificallydirected in particular to tissues of wheat seeds through geneexpression-regulating sequences such as, for example, the promoter of agene of γ gliadins or the promoter of the gene that codes for aD-hordein, one achieves post-transcriptional silencing of all of thegenes of the species soft wheat and hard wheat in an effective andsynergistic manner, as one is able to silence a greater number ofgliadin genes compared to the results of silencing of α and γ gliadinsby hpRNA described in the prior art. This is essentially due to thespecific design of the sense and antisense subsequences whose generatedsiRNA hybridizes with all of the mRNA of the α, β, γ, and ω gliadins ofwheat in combination with gliadin promoters having higher levels ofexpression that can be induced in specific tissues of the wheat seed.This design was arrived at by identifying groups of gliadins containingepitopes recognized by human T cells, such that silencing of theproteins containing them gives rise to wheat seeds that can be used toobtain products suitable for persons allergic to gluten.

In the present invention, the terms DNA and RNA are used to refer todeoxyribonucleic acid and ribonucleic acid respectively.

Therefore, one aspect of the present invention is a polynucleotide thatis at least 90% identical to a sequence comprising two sequence pairs(a1-a2) and (b2-b1) separated by a spacer sequence in which:

-   -   a. the sequences a1, a2, b1, and b2 differ among themselves and        are selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or        SEQ ID NO: 4, in the following form:    -   b. if a1 is SEQ ID NO: 1 or SEQ ID NO: 4, b1 is SEQ ID NO: 4 or        SEQ ID NO: 1, and a2 is SEQ ID NO: 2 or SEQ ID NO: 3, and    -   c. if a1 is SEQ ID NO: 2 or SEQ ID NO: 3, b1 is SEQ ID NO: 3 or        SEQ ID NO: 2, and a2 is SEQ ID NO: 1 or SEQ ID NO: 4.

Another aspect of the present invention is a polynucleotide thatcomprises two sequence pairs (a1-a2) and (b2-b1) separated by a spacersequence in which the sequences a1, a2, b1, and b2 are described inparagraphs (a) through (c) of the previous aspect.

The sequences a1-a2 and b2-b1 (referred to in the following as thesequence pairs of the invention) are linked to form a linear andcontinuous nucleotide sequence in which, in turn, the two pairs arelinked among themselves by means of a spacer sequence at least onenucleotide in length. Preferably, the spacer sequence is a non-codingsequence that is eliminated after the process of forming dsRNA. Thespacer sequence may be part of a sequence of an intron of a gene. Thefunction of the spacer sequence is to act as a hinge for the sequencepairs described so that pairing or hybridization of the RNA sequencescoding for the polynucleotide may take place.

The polynucleotide is at least 90% identical with the sequence thatcomprises the sequence pairs of the invention. In this manner one takesinto account changes in any of the nucleotides that make up the sequencepairs, up to a percentage of 90% identity with the original sequencewhose nucleotide composition is specified in the present aspect.

SEQ ID NO: 1 is the sense sequence that comprises a part of the fragmentthat codes for the epitopes of ω gliadins recognized by human T cellsthat give rise to an immune response in persons suffering from celiacdisease. SEQ ID NO: 2 is the sense sequence that comprises a part of thefragment that codes for the epitopes of α, β, and γ gliadins. SEQ ID NO:3 is the antisense sequence of SEQ ID NO: 2, and SEQ ID NO: 4 is theantisense sequence of SEQ ID NO: 1.

The polynucleotide of the invention gives rise to RNA in which the twosequence pairs hybridize with each other, forming a hairpin. Therefore,according to these first two aspects of the present invention, thecombinations of sequences by means of which RNA hairpins can be obtainedare shown in Table 1 and Table 2:

TABLE 1 Combinations of sequences in which the a1-a2 and b2-b1 pairs aresense or antisense sequences. Combinations a1 a2 b2 b1 1 SEQ ID NO: SEQID NO: SEQ ID NO: SEQ ID NO: 1 2 3 4 2 SEQ ID NO: SEQ ID NO: SEQ ID NO:SEQ ID NO: 2 1 4 3 3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 3 4 1 24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 4 3 2 1

TABLE 2 Combinations of sequences in which the a1-a2 and b2-b1 pairscontain sense and antisense sequences. Combinations a1 a2 b2 b1 1 SEQ IDNO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 1 3 2 4 2 SEQ ID NO: SEQ ID NO: SEQID NO: SEQ ID NO: 3 1 4 2 3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:2 4 1 3 4 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 4 2 3 1

In a preferred embodiment of the present invention, the polynucleotidecomprises two sequence pairs (a1-a2) and (b2-b1) separated by a spacersequence in which a1 is SEQ ID NO: 1, a2 is SEQ ID NO: 2, b2 is SEQ IDNO: 3, and b1 is SEQ ID NO: 4.

According to another preferred embodiment, the polynucleotide comprisestwo sequence pairs (a1-a2) and (b2-b1) in which the spacer sequence isSEQ ID NO: 5. The sequence SEQ ID NO: 5 is a fragment of an intron ofthe gene Ubi1 that codes for maize ubiquitin. An intron is a region ofDNA that is eliminated from the primary RNA transcript by a processreferred to as splicing, i.e., the intron does not code for any sequenceof a protein. Ubiquitin is a protein that has the function of markingother proteins for destruction.

Another preferred embodiment is a polynucleotide that also comprises agene expression-regulating sequence functionally linked to its 5′ end.In the present invention, the term gene expression-regulating sequencerefers to a nucleic acid sequence that affects the functionality of thegene with respect to the beginning of transcription of a DNA sequence orthe beginning of translation of an RNA sequence or other undescribedsequences. By way of example, the gene expression-regulating sequencescovered by the present invention are promoters and other less commonsequences such as certain introns. The regulatory sequence binds to the5′ end of the polynucleotide of the present invention in a functionalmanner, i.e., it is capable of directing the expression of thepolynucleotide with an intensity and localization that depend on its ownregulatory sequence.

According to a more preferable embodiment, the geneexpression-regulating sequence is SEQ ID NO: 6 and/or SEQ ID NO: 7. Thesequence SEQ ID NO: 6 corresponds to the sequence of a promoter of the γgliadin gene that shows a duplication in a proline box. SEQ ID NO: 7corresponds to the sequence of a promoter of the D-hordein gene (thesecond nucleotide of this gene has the accession number AY998009 andbelongs to the species Hordeum chilense). Both promoters are expressedin the endosperm of the seeds.

Also included are the sequences that are complementary to any of thepolynucleotides of the present invention.

In the following, the terms “polynucleotides of the invention” or“polynucleotides of the present invention” will be used to refer to anyof the above polynucleotides.

Another aspect of the present invention is an RNA sequence coded for byany of the polynucleotides of the invention and capable of forming anhpRNA in which the sequence coded for by the pair a1-a2 hybridizescompletely with the sequence coded for by the pair b2-b1.

An hpRNA (the English abbreviation for hairpin RNA) is a hairpin shapeformed by hybridization of the transcribed sequences. In the presentinvention, the polynucleotide of the present invention in which thesequence pairs a1-a2 and b2-b1 completely hybridize between themselveswas used as a template for synthesizing the transcribed sequences, ascan be seen in FIG. 1B. An hpRNA is a double-stranded RNA (dsRNA) thatis cleaved by an endoribonuclease, for example the endoribonucleaseDicer, resulting in fragments of approximately 21-25 nts. Thesefragments are known as siRNA. As has been described above, the siRNAscause post-transcriptional silencing of the target nucleotide sequencesso that the protein that would result from the expression of mRNAsequences is not obtained.

Another aspect of the present invention is at least one siRNA generatedfrom the sequence of the hpRNA according to the previous aspect. ThesiRNA can also be referred to as RNAi. The siRNA is a double-strandedRNA of between 21 and 25 nucleotides, but is not limited to this numberof nucleotides, and it is generated from the hpRNA sequence of theinvention. In the present invention, in defining the approximate numberof nucleotides of the siRNA (approximately 21 and 25 nucleotides), it isunderstood that there is another strand that is complementary to thissequence, i.e., that one can use the terms nucleotides or base pairs(bp) interchangeably.

As has been described, the Dicer enzyme cleaves the dsRNA intodouble-stranded fragments of approximately 21-25 nucleotides (siRNA),with the 5′ end phosphorylated and two unpaired nucleotides protrudingat the 3′ end. Of the two strands of siRNA, only one, referred to as theguide strand, is incorporated into the enzymatic complex RISC, while theother is degraded. The thermodynamic characteristics of the 5′ end ofthe siRNA determine which of the two strands is incorporated into theRISC complex. The strand that is less stable at the 5′ end is normallyincorporated as the guide strand. The guide strand must be complementaryto the mRNA that is to be silenced in order for post-transcriptionalsilencing to occur. Subsequently, the RISC complex binds to thecomplementary mRNA of the guide strand of the siRNA present in thecomplex, and cleavage of the mRNA occurs.

Another aspect of the present invention is an expression vector thatcomprises any of the polynucleotides of the invention. In the following,“vector of the invention” or “vector of the present invention.”

The term “vector” refers to a DNA fragment that has the capacity toreplicate itself in a particular host, and, as the term indicates, mayserve as a vehicle for multiplying another DNA fragment that has beenfused to it (an insert). “Insert” refers to a DNA fragment that is fusedto the vector; in the case of the present invention, the vectorcomprises the polynucleotide of the invention, which, when fusedthereto, can replicate itself in the corresponding host. Vectors may beplasmids, cosmids, bacteriophages, or viral vectors, without excludingother types of vectors that meet the present definition of vector.

Another aspect of the present invention is an isolated cell transfectedwith the vector of the invention. In the following, “cell of theinvention” or “cell of the present invention.” The term “cell” as usedin the present invention refers to a prokaryotic or eukaryotic cell. Thecell may be a bacterium capable of replicating foreign DNA bytransforming, for example, any of the strains of the species Escherichiacoli or a bacterium capable of transferring the DNA of interest into theinterior of a plant, such as for example Agrobacterium tumefaciens.Preferably, the cell refers to a eukaryotic plant cell, and within thisgroup, more preferably to cells belonging to the kingdom Plantae.Therefore, in cases in which the cell is a plant cell, the term “cell”comprises at least a parenchyma cell, meristem cell, or a cell of anytype, differentiated or undifferentiated. Thus, this definition alsoincludes a protoplast (a plant cell lacking a cell wall).

The term “transfection” refers to the introduction of external geneticmaterial into cells via plasmids, viral vectors (in this case one canalso use the term “transduction”), or other means of transfer. The term“transfection by nonviral methods” is used with reference to mammalianeukaryotic cells, while the term “transformation” is preferred todescribe nonviral transfers of genetic material into bacteria andnon-animal eukaryotic cells such as yeasts, algae, and plants. In thecase of the present invention, the term “transfection” is equivalent tothe term “transformation.”

Another aspect of the present invention is a genetically modified plantthat comprises the cell of the invention. The term “plant” includesevery part of the plant, which may be preserved or cultivated eitherindividually or in combination, as well as the germplasm. The germplasmis composed of biological material that contains interspecies geneticvariability or the genetic materials that can perpetuate a species orpopulation of an organism (see seeds, propagule, or progeny below). Theplant must comprise the cell of the present invention in a form that isexpressed in a specific tissue (at a specific moment of plantdevelopment or depending on the environmental conditions in which itdevelops) or in a constitutive or epitopic form (expressed in othercells or tissues differing from those that are common and expected).

The plant of the invention may contain the polynucleotide of theinvention in homozygosis, heterozygosis, or hemizygosis.

According to a preferred embodiment, the plant belongs to the genusTriticum. The plant is selected from the list that includes, but is notlimited to, Triticum aestivum, T. aethiopicum, T. araraticum, T.boeoticum, T. carthlicum, T. compactum, T. dicoccoides, T. dicoccum, T.durum, T. ispahanicum, T. karamyschevii, T. macha, T. militinae, T.monococcum, T. polonicum, T. repens, T. spelta, T. sphaerococcum, T.timopheevii, T. turanicum, T. turgidum, T. urartu, T. vavilovii and T.zhukovskyi.

According to another preferred embodiment, the plant is of the speciesTriticum aestivum or Triticum turgidum. According to another preferredembodiment, the plant belongs to the cultivar Bobwhite or the cultivarDon Pedro. More preferably, the cultivars BW208 and BW2003 (Bobwhite),which belong to the wheat species Triticum aestivum L. ssp aestivum, andthe variety Don Pedro, which belongs to the wheat species Triticumturgidum L. ssp durum, are selected.

Bobwhite is the name of the cultivar obtained from the InternationalMaize and Wheat Improvement Center (CIMMYT). BW208 and BW2003 aredifferent Bobwhite lines. Don Pedro is a hard wheat variety, also fromCIMMYT. Bobwhite and Don Pedro are public varieties.

The plant of the invention may be obtained by genetic transformation ofplant cells by means of biolistics, Agrobacterium tumefaciens, or anyother technique that allows integration of the polynucleotide of theinvention into the DNA of the plant, whether this DNA be genomic,chloroplastic, or mitochondrial, followed, although not necessarily, byan in vitro regeneration program suited to the characteristics andrequirements of the transformed plant species. Moreover, the plant mayalso be obtained by transference of any of the sequences of theinvention by crossing, e.g., using pollen of the plant of the inventionto pollinate any other plant that does not contain the polynucleotide ofthe invention, or pollinating the gynoecia of plants containing thepolynucleotide of the invention with other pollen that does not containthese sequences. The methods for obtaining the plant of the inventionare not exclusively limited to those described in this paragraph; forexample, genetic transformation of germ cells from the ear of wheatcould be carried out as mentioned, but without having to regenerate aplant afterward (see below). Moreover, a plant that comprises the cellof the present invention in a stable or transient form is also included.

Another aspect of the present invention is a seed from any of the plantsof the invention. This will be referred to below as the “seed of theinvention” or “seed of the present invention”.

Another aspect of the present invention is a grain of pollen, propagule,progeny, or plant part derived from any of the plants of the invention.

In the present invention, pollen is taken into account as a transmitterof the genetic and phenotypic characteristics that may result frompollination of any plant variety compatible with the pollen in question.In this manner, one obtains a plant that comprises the polynucleotide ofthe present invention, and, after the respective crosses and/orselections, one can obtain a plant in which the sequence is integratedin stable form (although it may also be expressed in transient form) andin a sufficient number of copies to obtain the same desirablecharacteristics in subsequent generations.

Propagules are parts of the plant that allow asexual propagation orreproduction in plants, whereby new individualized plants or organs areobtained. The tissues of the separated portion must recover to thestatus of meristems in order to produce the entire group of organs ofthe plant. The propagule is selected from the list that comprises,without being exhaustive, stolons, rhizomes, tubers, or bulbs.

The term “progeny” refers to the result of reproduction, i.e., theindividual or individuals produced by the intervention of one or moreparent individuals. For example, the progeny of plants obtained bysexual reproduction are seeds, but the progeny of a plant may be anycell resulting form the fusion of any cellular contents, plastid,cellular compartment, DNA, or any combinations thereof. In the processesof cellular division (such as in vitro cultivation, for example), theprogeny are the cells resulting from the division.

Another aspect of the present invention is the use of thepolynucleotide, vector, or cell of the invention for the silencing ofalpha, beta, gamma, and omega gliadins of Triticum spp.

As shown in Example 2 of the present invention, the integration of thepolynucleotide of the invention into the genome of wheat plants of twogenotypes of wheat for flour (Triticum aestivum L.), cultivar Bobwhite(BW208 and BW2003), and one of hard wheat (Triticum turgidum ssp durum),cultivar Don Pedro, causes silencing of the alpha, beta, gamma, andomega gliadins of the seeds of transformant plants (FIG. 3, FIG. 4, andFIG. 5).

Another aspect of the present invention is the use of the seed of theinvention to prepare a food composition (referred to in the following asthe “composition of the invention” or “composition of the presentinvention”). The food composition is prepared from, but not limited to,the flour and/or semolina of the seeds of the invention, combined or notwith other flours and/or semolinas, or other compounds.

The term “flour” as it is understood in the present invention refers tothe product obtained by milling of any seed or plants of the genusTriticum, with the bran or husk of the seed removed to a greater orlesser degree.

The term “semolina” refers to coarse flour (slightly milled wheatseeds), i.e., fragments of the endosperm with a variable amount of seedhusks.

The prepared food is selected from, but not limited to, the listcomprising bread, bakery products, pastries, confectionery products,food pasta, food dough, grains, drinks, or dairy products.

Another aspect of the invention is use of the composition of theinvention to prepare a functional food product, vitamin supplement, ornutritional supplement. As understood in the present invention, a foodproduct fulfills a specific function, such as improving the diet ofthose who consume it. For this purpose, a vitamin and/or nutritionalsupplement may be added to the functional food product.

The food product that comprises the food composition of the presentinvention may be eaten even by persons who are allergic to gluten, i.e.,suffer from celiac disease.

Another aspect of the present invention is a method for obtaining theplant of the invention, comprising the following:

-   -   a. selecting a part of the plant,    -   b. transfecting the cells of the part of the plant of        paragraph (a) with a vector according to claim 9,    -   c. selecting the transfected cell of paragraph (b) that        comprises the polynucleotide according to any of claims 1        through 6,    -   d. regenerating at least one plant derived from the cell        selected in paragraph (c),    -   e. selecting one or more plants regenerated according to        paragraph (d) in which the polynucleotide is transcribed into an        hpRNA, and    -   f. selecting one or more plants obtained according to        paragraph (e) that show silencing of the alpha, beta, gamma, and        omega gliadins in its seeds.

In the case of wheat plants, one should preferably select the scutellumto be transfected by the vector of the invention. The insertion of thepolynucleotide of the present invention into a vector may be carried outby cloning methods that are known in the art, by means of cleaving thepolynucleotide and the vector with restriction enzymes (digestion) andsubsequent ligation, such that the sequence of the vector comprises thepolynucleotide of the invention. The vector was described in a previousparagraph.

The selection of the vector that comprises the selected sequence of theinvention may be carried out by techniques such as the following:

-   -   Selection of cells containing the vectors of the invention by        means of adding antibiotics to the culture medium. The        resistance of these cells to substances such as antibiotics is        produced by the synthesis of molecules coded for by a sequence        contained in the sequence of the vector.    -   Digestion with restriction enzymes, by means of which one        obtains a fragment of one of the sequences of the invention        inserted into the vector.

The cell is obtained by any type of microbiological culture (forexample, E. coli or Agrobacterium tumefaciens) or plant culture.

Genetic transformation of the cells is carried out using techniquesknown in the art, such as, for example, electroporation, genetictransformation by biolistics, Agrobacterium tumefaciens, or any othertechnique that allows the integration of any of the sequences of theinvention into the DNA of the cell. Preferably, transformation should becarried out by biolistics. By means of these techniques, one can obtainin a stable manner a vector that comprises any of the sequences of theinvention, such that after successive cell divisions, the incorporatedsequence continues to express itself. Cells including any of thesequences of the invention in a transient manner are also included.

The cell transformed with a vector that comprises any of thepolynucleotides of the invention may incorporate the sequence in anytype of cellular DNA: nuclear, mitochondrial, and/or chloroplastic, andin this case, one usually inserts the DNA, which comprises, among othersequences, the polynucleotide of the invention. Selection of cells thathave incorporated any of the sequences of the invention is carried outby adding antibiotics to the culture medium that provides nutrients tothem. The resistance of these cells to substances such as antibiotics orherbicides is produced by the synthesis of molecules coded for by asequence contained in the DNA sequence of the vector. One may alsoselect the cell that comprises the polynucleotide of the invention byany other technique that allows its presence or absence and/or itsexpression to be distinguished.

The plant cells selected may be subjected to a program of organogenesisor somatic embryogenesis, thus giving rise to a complete plant thatcomprises the genetic material of the original cell from which itoriginated. This is possible because of the fact that plant cells aretotipotent, i.e., by means of a suitable combination of hormones, theycan be dedifferentiated, thus generating embryonic cells that, becausethey contain a complete copy of the genetic material of the plant towhich they belong, have the potential to regenerate a complete newplant. Light and temperature conditions suited to each plant species arealso required. Once the plant originating from the selected plant cellhas regenerated itself, one can carry out an analysis of the presenceand/or expression of the nucleotide sequence that codes for thepolynucleotide of the invention or any other sequence of the presentinvention (promoter sequence, etc.)

The method also includes the selection of a plant that shows substantialsilencing of the alpha, beta, gamma, and omega gliadins in its seeds.Preferably, plants showing virtually complete or complete silencing ofall the gliadins of the seeds should be selected. The reduction in totalgliadin content of a control plant (a plant not including thepolynucleotide of the invention) is greater than or equal to 90%. Thecontrol plants preferably do not contain the polynucleotide of theinvention in the plant cell. Prior to being transformed, the control mayalso be a wild-type plant that has undergone the same in vitrocultivation steps as the plants of the invention or has not undergonethese cultivation steps.

The transfected cells may be germ cells from the ear of the plant, andin this case, at least one plant derived from the seeds generated bysaid ear of the plant would be regenerated, and one would select atleast one plant showing silencing of the alpha, beta, gamma, and omegagliadins in its seeds.

Throughout the description and the claims, the word “comprise” and itsvariants is not intended to exclude other technical characteristics,additives, components, or steps. For the person skilled in the art,other objects, advantages, and characteristics of the invention will beobvious partly from the description and partly from the practice of theinvention. The following figures and examples are provided by way ofillustration, and they are not intended to limit the scope of thepresent invention.

DESCRIPTION OF THE FIGURES

FIG. 1. Shows the structure of the polynucleotide of the invention.

Figure A shows a polynucleotide that comprises the sequence pairs a1-a2and b2-b1, separated by a spacer sequence (E) whose expression isdirected by a gene expression-regulating sequence (R).

Figure B shows the hpRNA resulting from transcription of thepolynucleotide represented in Figure A in which is formed a hairpin, inwhich the sequences Sec a1, a2, b2, and b1 hybridize as is described.Subsequently, this RNA is processed, producing a new sequence ofdouble-stranded RNA (dsRNA). The final step shown refers tofractionation of the prior sequence by enzymes such as, for example, theenzyme Dicer, such that double-stranded RNA sequences of approximately21-25 nucleotides, referred to as siRNA (small interfering RNA), areformed.

FIG. 2. Shows a specific example of the polynucleotide of the invention.

In this case, the sequence R is a promoter sequence of the gene ofgamma-gliadin (γ gliadin) or D-hordein. Sequence E is a fragment of anintron of the maize ubiquitin gene. NOSt is a transcription terminationsequence. This polynucleotide is inserted in this specific example intoa pUC18 vector that has an ampicillin resistance gene.

FIG. 3. Shows separation and identification of gliadins of plant seedsof the wheat variety BW208, transformed with the polynucleotide of theinvention, by the A-PAGE and MALDI-TOF techniques.

Figure A shows separation by A-PAGE of the gliadins of the sample ofseeds of a control line of wheat for flour (Triticum aestivum L. ssp.aestivum), variety BW208, and a line of wheat for flour of the samegenotype transformed with the vector pGhp-ω/α/β/γ (B377-2-3). The groupsof gliadins obtained by separation are indicated.

Figure B shows a MALDI-TOF analysis of gliadins from a sample of acontrol line of seeds from wheat for flour (Triticum aestivum L. ssp.aestivum), variety BW208, and a line of wheat for flour of the samegenotype transformed with the vector pGhp-ω/α/β/γ. In the diagramcorresponding to the control line, the various fractions of alpha (α),beta (β), gamma (γ), and omega (ω)-gliadins are indicated. The X axisrepresents the ratio (m/z) of the mass of given ion (m) and the numberof protons it contains (z).

FIG. 4. Shows separation and identification by A-PAGE of the gliadins ofplant seeds of the wheat variety BW2003 transformed with thepolynucleotide of the invention.

A-PAGE gel separation of gliadins from a control line of wheat for flour(Triticum aestivum L. ssp. aestivum), variety BW208, and a line of wheatfor flour of the same genotype transformed with the vector pGhp-ω/α/β/γ.The groups of gliadins obtained in the separation are indicated.

FIG. 5. Shows separation and identification by the A-PAGE technique ofgliadins of seeds from plants of the hard wheat variety Don Pedrotransformed with the polynucleotide of the invention.

A-PAGE gel separation of gliadins of a control line of hard wheat(Triticum turgidum L. ssp. durum), variety Don Pedro, and a line of hardwheat of the same genotype transformed with the vector pGhp-ω/α/β/γ. Thegroups of gliadins obtained in the separation are indicated.

FIG. 6. Shows separation and identification by the A-PAGE technique ofgliadins of seeds from plants of the variety BW208 transformed with thepolynucleotide of the invention.

A-PAGE gel separation of gliadins of a control line BW208 and threeseeds of a line (B382-4-1) of the same genotype transformed with thevector pDhp-ω/α/β/γ. The groups of gliadins obtained by separation areindicated. Note that the promoter that comprises this vector is aD-hordein promoter (SEQ ID NO: 7).

FIG. 7. Shows western blot analysis of two lines of wheat for flour(Triticum aestivum L.) transformed with the vector pDhp-ω/α/β/γ.

A: The gliadins of three grains of the line B382-4-1, denoted I1, I2,and I3, of the genotype BW208 were subjected to SDS gel separation andhybridized with the gluten-specific monoclonal antibody R5.

B: The gliadins of two grains of the line B374-6-2, denoted O2 and O3,of the genotype BW2003 were subjected to SDS gel separation andhybridized with the gluten-specific monoclonal antibody R5.

The numbers on the left in A indicate molecular weight in kDa, both forA and for B.

FIG. 8. Shows western blot analysis of two lines of wheat for flour(Triticum aestivum L.) of the variety BW208 transformed with the vectorpGhp-ω/α/β/γ.

The gliadins of three grains of the transgenic line B375-3-1, denotedA1, A2, and A3, and the gliadins of two grains of wheat of thetransgenic line B377-2-3, denoted C1 and C2, both of the genotype BW208,were subjected to SDS gel separation and hybridized with thegluten-specific monoclonal antibody R5.

The numbers on the left in the figure indicate molecular weight in kDa.

EXAMPLES

In the following, the invention will be illustrated by means of severaltests conducted by the inventors that describe the construction of thepolynucleotide of the invention, the generation of wheat plants of 3distinct varieties transformed with the vector of the invention, andanalysis of gliadin content using the techniques A-PAGE and MALDI-TOF.

Example 1. Construction of the Vectors pGhp-ω/α/β/γ and pDhp-ω/α/β/γ

1.1 Synthesis of α/β/γ and ω Gliadin Sequences.

The DNA sequences deposited in the Genebank belonging to wheat α/β/γ andω gliadins were aligned separately, and the regions showing the greatestdegree of homology were identified. Based on these alignments, weselected a 170 bp sequence of α/β/γ gliadins and another 191 bp sequenceof ω gliadins and designed the primers Alpha_hp-F (SEQ ID NO: 8) andAlpha_hp-R (SEQ ID NO: 9) for amplification of the α/β/γ fragment andthe primers Omega_III-F (SEQ ID NO: 10) and Omega_III-R (SEQ ID NO: 11)for amplification of the ω fragment (Table 1). The PCR conditions forthe two fragments were as follows: cDNA of T. aestivum cv Bobwhitesynthesized from 50 ng of total RNA extracted from immature grains, 1.5mM of MgCl₂, 0.2 mM of dNTPs, 0.2 μM of each primer, 1× buffer, and0.625 units of a mixture of polymerases in a 100:1 ratio of Tth (Thermusthermophilus) to Pfu (Pyrococcus furiosus) (BIOTOOLS, Madrid, Spain) ina final reaction of 25 μl. The conditions of the PCR cycles were asfollows: an initial pass of 94° C. 5 min, 35 cycles of 94° C. 30 sec,55° C. 30 sec, and 72° C. 30 sec; and a final extension of 72° C. 4 minin a GeneAmp PCR system 9700 thermocycler (Applied Biosystems). Theproducts of each PCR were purified using the GFX PCR DNA PurificationKit (Amersham Biosciences, Amersham, UK) and were sequenced by the firmSecugen SL. In order to achieve overlapping of the α/β and ω fragments,the ω gliadin fragment was again amplified using an overlapping primer(overlapping Omega III R (SEQ ID NO: 12) together with the direct primerSEQ ID NO: 10), and the α/β gliadin fragment was again amplified usinganother overlapping primer, overlapping Alpha F (SEQ ID NO: 13),together with the reverse primer SEQ ID NO: 9) which added to eachfragment 12 base pairs that were complementary with the other fragment.By means of the latter amplifications, we obtained two fragments thatcomplemented each other between the 3′ end of the ω gliadin fragment andthe 5′ end of the α/β/γ gliadin fragment. The PCR conditions were thesame as described previously; the product of these reactions wasseparated in 1% agarose gel and the band corresponding to each fragmentwas purified with the QUIAquick Gel Extraction Kit (QUIAGEN Inc.,Valencia, Calif.). The final overlapping PCR was carried out using 10 ngof the α/β/γ purified overlapping fragment, 10 ng of the purified cofragment, 1.5 mM of MgCl₂, 0.2 mM of dNTPs, 0.2 μM of the primeralpha_hp-F, 0.2 μM of the primer omega_III-R, 1× buffer, and 0.625 unitsof a 100:1 mixture of Tth/Pfu polymerases in a final reaction of 50 μl.The conditions of the PCR cycles were as follows: an initial pass of 94°C. 2 min, 35 cycles of 94° C. 30 sec, 57° C. 30 sec, and 72° C. 30 sec;and a final extension of 72° C. 4 min in a GeneAmp PCR system 9700thermocycler (Applied Biosystems). The product of these PCR wasseparated in 1% agarose gel and the band showing the size correspondingto the ω/α/β (361 bp) fragment was purified with the QUIAquick GelExtraction Kit (QUIAGEN Inc., Valencia, Calif.) and cloned in theplasmid TOPO (Invitrogen, Carlsbad, Calif.). This plasmid comprises thesites attL1 and attL2 that allow transference through recombination ofthe gene of interest into any Gateway® vector that comprises the sitesattR1 and attR2.

1.2 Obtaining the Transformation Vectors pGhp-ω/α/β/γ and pDhp-ω/α/β/γ.

The transformation vector was synthesized using the vector puc18 (2616bp). The various fragments that comprise the transformation vectorpGhp-ω/α/β/γ were introduced one by one into the multiple cloning siteof the vector puc18. The fragment Nost (272 bp) was extracted from thevector pANDA-β by restriction with the enzyme EcoRI and introduced intothe puc18, giving rise to puc18_Nost. Next, the fragment attRsense GUSattRantisense (4.4 kb) of the vector pANDA was extracted by restrictioncombined with the enzymes Sacl and Kpnl and introduced into the vectorpuc18_Nost, giving rise to puc18_attR_GUS_Nost. This fragment containedthe sites attR1 and attR2 with sense and antisense sequences separatedby a linking segment of 1 kb (gus linker). This gus linker sequence wassubstituted for the Ubi intron fragment (1019 bp) previously cloned inour laboratory by restriction with the enzyme EcoRV, producing theplasmid puc18_attR_Ubi_Nost. Finally the gliadin promoter (885 bp) wasintroduced by double restriction with the enzymes Sphl and Xhol,producing the plasmid puc18_Gli_attR_Ubi_Nost.

The D-hordein gene promoter (836 bp) was also introduced by doublerestriction with the enzymes Sphl and Xhol, producing the vectorpuc18_D_attR_Ubi_Nost. The difference between the vectors pGhp-ω/α/β/γand pDhp-ω/α/β/γ is that the former contains the promoter of wheat γgliadins and the latter contains the promoter of wheat H-hordein.

The next step was the introduction of the fragment ω/α/β into theplasmids puc18_Gli_attR_Ubi_Nost and puc18_D_attR_Ubi_Nost. The plasmidTOPO+ ω/α/β contained the sites attL1 and attL2, while the plasmidspuc18_Gli_attR_Ubi_NOSt and puc18_D_attR_Ubi_Nost contained the sitesattR1 and attR2 with sense and antisense sequences separated by theintron Ubi. This made it possible to carry out an LR recombinationreaction using the kit Gateway® LR Clonase™ Enzyme Mix (Invitrogen,Carlsbad, Calif.) and following the manufacturer's instructions. Theresult was the introduction of the fragment ω/α/β/γ having sense andantisense sequences separated by the intron Ubi into the structure ofthe plasmids puc18_Gli_attR_Ubi_NOSt and puc18_D_attR_Ubi_Nost. Theresulting vectors were designated pGhp-ω/α/β/γ and pDhp-ω/α/β/γ (FIG. 2)and were introduced by transformation into competent E. coli cells(DH5α) for their subsequent multiplication.

Example 2. Obtaining Transgenic Wheat Lines

Genetic transformation was carried out by biolistics using a system foraccelerating particles with pressurized helium (PDS1000/He™. BIORAD,Hercules, Calif.). We used two genotypes of wheat for flour (Triticumaestivum L.) cultivar Bobwhite (BW208 and BW2003) and one of hard wheat(Triticum turgidum ssp durum) cultivar Don Pedro to isolate the scutellaof immature embryos. Isolation was carried out in a sterile environmentusing immature wheat grains collected 12-16 days after anthesis,previously sterilized by immersion for 3 min in a 70% ethanol solution,10 min in a 20% sodium hypochlorite solution, and rinsing twice withsterile distilled H₂O. For genetic transformation, we used goldparticles 0.6 μm in diameter and mixed in 1.5 pmoles/mg gold of thevector pGhp-ω/α/β/γ or the vector pDhp-ω/α/β/γ and 0.5 pmoles/mg of thevector pAHC25 (Christensen et al., 1996. Transgenic Research 5,213-218). The conditions of each shot were as follows: 91.4 kPa (27inHg) vacuum pressure, 7.584 MPa (1100 PSI) shot pressure, 6 cm shotdistance, and 60 μg of the mixture of gold and plasmids per shot.

Cotransformation with the plasmid pAHC25, which contains the barselection gene (resistance to phosphinothricin) and the uidA gene(synthesis of β-glucuronidase), allowed selection of the tissuestransformed in media with 4 mg/l of phosphinothricin (PPT) andsubsequent identification of transgenic tissues by means of theβ-glucuronidase assay (GUS) in accordance with the protocol described byJefferson (1987, Plant Mol Biol Rep 5:387-405). The media, the in vitrocultivation process, and regeneration of the plants were in accordancewith Barro et al (Barro et al., 1998. Theoretical and Applied Genetics97, 684-695).

The plants regenerated in in vitro cultivation were placed in the soil,and they were then subjected to the GUS assay per se and as described inthe previous paragraph. From the plants that yielded a positive resultin the GUS assay, DNA was extracted using DNAzol reagent (Invitrogen,Carlsbad, Calif.) following the manufacturer's instructions, and PCR wascarried out to confirm the presence of the plasmids pGhp-ω/α/β/γ,pDhp-ω/α/β/γ, and pAHC25 in the genome of the adult plant. PCRconditions were as follows: 100 ng of DNA extracted from young leaves,1.5 mM MgCl₂, 0.2 mM dNTPs, 0.2 μM of each primer, 1× buffer, and 0.625units of polymerase Tth (BIOTOOLS, Madrid, Spain). The primers used wereprGliF (SEQ ID NO: 14) and overlapping Omega_III_R (SEQ ID NO: 12) forthe plasmid pGhp-ω/α/β/γ, prHorDF (SEQ ID NO: 15) and overlappingOmega_III_R (SEQ ID NO: 12) for the plasmid pDhp-ω/α/β/γ and BAR_F (SEQID NO: 16) and BAR_R (SEQ ID NO: 17) for pAHC25 (Table 3). Theconditions of the PCR cycles were as follows: an initial pass of 94° C.5 min, 35 cycles of 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 sec; anda final extension of 72° C. 7 min in a GeneAmp PCR system 9700thermocycler (Applied Biosystems). The product of each PCR was separatedby electrophoresis in 1% agarose gel, and the positive lines wereselected for subsequent analysis.

TABLE 3 Primers and sequences used. Primer 5′ to 3′ sequence Alpha_hp_F(SEQ ID NO: 8) Alpha_hp_R (SEQ ID NO: 9) Omega_III_F (SEQ ID NO: 10)Omega_III_R (SEQ ID NO: 11) Overlapping (SEQ ID NO: 12) Omega_III_ROverlapping Alpha_F_ (SEQ ID NO: 13) prGli_F (SEQ ID NO: 14) prHorDF(SEQ ID NO: 15) BAR_F (SEQ ID NO: 16) BAR_R (SEQ ID NO: 17)

Example 2. Extraction of Gliadins and MALDI/TOF and A-PAGE Analysis

The seeds of the positive lines were crushed in a mortar to obtainflour. After this, the flour was washed with 1 ml of a 0.5 M NaClsolution for 15 minutes at room temperature (RT) in an agitator andcentrifuged at 13,000 rpm for 10 min. The supernatant was discarded, andthe precipitate was washed with distilled water for 15 min at RT, alsounder agitation. After this, the samples were centrifuged at 13,000 rpmfor 10 min, after which the supernatant was discarded. The gliadins wereextracted from the precipitate with 60% (v/v) aqueous ethanol solutionin a 5:1 ratio (μl ethanol:mg flour) and agitation was carried out for45 min at RT. The samples were then centrifuged at 13,000 rpm and thesupernatant containing the gliadins was collected in fresh tubes. Afraction of the extract was used for identification of the gliadins byMALDI-TOF mass spectrometry, and another fraction was separated byacidic polyacrylamide gel electrophoresis (A-PAGE). For the MALDIanalysis, we took 5 μl of the gliadin extracted and added to this 2 μlof a 50 mM octyl-β-D-glucopyranoside (ODGP) solution and 25 μl ofsinapic acid saturated in a 30% (v/v) aqueous acetonitrile solutioncontaining 0.1% (v/v) of trifluoroacetic acid (TFA), used as a matrixsolution. The mixture was dried in a Speed-Vac centrifuge for 15 min,and the residue was dissolved in 6 μl of a 60% (v/v) aqueous ethanolsolution containing 0.1% TFA. The mixture was placed in a stainlesssteel sample holder and allowed to dry for 5 min at RT. The samples wereanalyzed in a MALDI-TOF Voyager DE-PRO (PE Biosystems) in the standardinstrument configuration. Spectra were recorded in linear positive modeat a voltage acceleration of 25 kV with a voltage grid of 93% and adelay of 700 nanoseconds. 200 laser spectra were accumulated toconstruct the gliadin profiles.

The gliadins were subjected to gel separation by A-PAGE following thestandard protocols described by Khan et al. (1985. Cereal Chemistry 62:310-313).

Hybridization (western blot) of the gliadins subjected to gel separationby SDS-PAGE was carried out with the antibody R5 (Valdez et al. 2003,Eur. J. Gastroenterol. Hepatol. 15:465-474), as this is the officialmethod recognized by the Codex Alimentarius for the detection of glutenin food.

The A-PAGE and MALDI-TOF gel assays showed that the combination of thishybrid sequence is highly effective in silencing wheat gliadins.

FIG. 3 shows separation and identification of gliadins of plant seeds ofthe wheat cultivar BW208 transformed with the polynucleotide of theinvention by means of the A-PAGE and MALDI-TOF techniques. In FIG. 3A,one observes band attenuations corresponding to each of the alpha, beta,gamma, and omega gliadins derived from seeds of a line of wheat forflour transformed with the vector pGhp-ω/α/β/γ (B377-2-3) compared tothe wheat cultivar BW208 from which it is derived (Triticum aestivum L.ssp. aestivum). In FIG. 3B, one observes the spectral expression profilecorresponding to the alpha, beta, gamma, and omega gliadins, with eachof the peaks corresponding to a different protein. In the diagram, thepeaks corresponding to each type of gliadin are indicated. As can beobserved in the expression profiles corresponding to the control plantsand those of the plants transformed with the polynucleotide of theinvention, the suppression of the peaks corresponding to each of thegliadins observed in the profile of the control plants is quitedistinct, thus demonstrating the efficacy of the polynucleotide andmethod of the present invention in post-transcriptional silencing of thegliadins present in the grains of wheat belonging to this cultivar.

FIGS. 4 and 5 show the separation and identification of gliadins ofplant seeds of the wheat cultivar BW2003 (Triticum aestivum L. ssp.aestivum) and the variety Don Pedro (Triticum turgidum L. ssp durum)respectively transformed with the polynucleotide of the invention bymeans of the A-PAGE technique. In both cases, one can see the bandattenuation corresponding to each of the alpha, beta, gamma, and omegagliadins derived from seeds of the wheat lines transformed with thevector pGhp-ω/α/β/γ (B377-2-3) compared to their respective controls.

FIG. 6 shows A-PAGE gliadin gel separation of a control line BW208 andthree seeds of a line (B382-4-1) of the same genotype transformed withthe vector pDhp-ω/α/β/γ. The promoter that contains this vector is thepromoter of a gene D-hordein (SEQ ID NO: 7). One can see the bandattenuation in the seeds of the transgenic lines corresponding to eachof the alpha, beta, gamma, and omega gliadins of wheat.

FIG. 7 shows a western blot analysis of two lines of wheat for flour(Triticum aestivum L. ssp. aestivum) transformed with the vectorpDhp-ω/α/β/γ that contain the promoter coded for by the sequence SEQ IDNO: 7.

In gel A, one can see the SDS-PAGE gel separation of the gliadins ofthree wheat grains (I1, I2, and I3) of the transgenic line B382-4-1. Ingel B, one can see the separation of the gliadins of two wheat grains(O2 and O3) of the transgenic line B374-6-2. After this, both gelsunderwent hybridization with the monoclonal antibody R5, whichrecognizes peptides that are potentially toxic for celiac patients.Monoclonal antibody R5 is the method officially recognized by the CodexAlimentarius for the detection of gluten in food.

In FIG. 7A, one does not observe an appreciable level of gliadins in theBW208 wheat lines that express the peptide of the invention, which isexpected for this type of detection with the antibody R5. In FIG. 7B,one can see a considerable reduction of gliadins in this othertransgenic wheat variety BW2003.

In FIG. 8, one sees on an SDS-PAGE gel the attenuation of the gliadinsof the three grains of the transgenic line B375-3-1, designated A1, A2,A3, and the gliadins of the two wheat grains of the transgenic lineB377-2-3, both of the genotype BW208, when they hybridize with the R5gluten-specific antibodies. The transgenic lines contain the promoter ofγ gliadins, i.e., they are transformed with the vector pGhp-ω/α/β/γ.

The invention claimed is:
 1. A polynucleotide comprising a firstsequence pair that is identical to a sequence pair designated (a1-a2), asecond sequence pair that is identical to a sequence pair designated(b2-b1), and a spacer sequence separating the first sequence pair andthe second sequence pair, wherein: (a) the sequences a1, a2, b1, and b2differ among themselves and are selected from SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, and SEQ ID NO:4, in the following form: (b) if a1 is SEQ IDNO:1 or SEQ ID NO:4, then b1 is SEQ ID NO:4 or SEQ ID NO:1, and if a2 isSEQ ID NO:2 or SEQ ID NO:3, then b2 is SEQ ID NO:3 or SEQ ID NO:2; (c)if a1 is SEQ ID NO:2 or SEQ ID NO:3, then b1 is SEQ ID NO:3 or SEQ IDNO:2, and if a2 is SEQ ID NO:1 or SEQ ID NO:4, then b2 is SEQ ID NO:4 orSEQ ID NO:1; and (d) when contained within a plant cell, the sequence ofthe polynucleotide is transcribed into RNA in which a first RNA sequencetranscribed from the sequence pair designated (a1-a2) hybridizes with asecond RNA sequence transcribed from the sequence pair designated(b2-b1) to form a double-stranded RNA (dsRNA) that is processed withinthe plant cell to generate siRNAs that reduce the levels of alpha, beta,gamma and omega gliadins in the plant cell.
 2. The polynucleotide ofclaim 1, wherein a1 is SEQ ID NO:1, a2 is SEQ ID NO:2, b2 is SEQ IDNO:3, and b1 is SEQ ID NO:4.
 3. The polynucleotide of claim 1, whereinthe spacer sequence is SEQ ID NO:5.
 4. The polynucleotide of claim 1,further comprising a gene expression-regulating sequence functionallylinked to the 5′ end of the polynucleotide, wherein the geneexpression-regulating sequence is SEQ ID NO:6 and/or SEQ ID NO:7.
 5. AnRNA sequence encoded by the polynucleotide of claim 1, wherein the RNAsequence forms an hpRNA in which the sequence coded for by the paira1-a2 completely hybridizes with the sequence coded for by the pairb2-b1.
 6. An siRNA generated from the sequence of the hpRNA encoded bythe RNA sequence of claim
 5. 7. An expression vector comprising thepolynucleotide of claim
 1. 8. An isolated cell transfected with theexpression vector of claim
 7. 9. A method for silencing alpha, beta,gamma, and omega gliadins of Triticum spp, the method comprisingproviding a plant of the genus Triticum and administering to the plantexternal genetic material that reduces the expression levels of thegenes encoding alpha, beta, gamma and omega gliadins, wherein theexternal genetic material targets a nucleotide sequence defined by SEQID NO:2 or a sequence complementary thereto (SEQ ID NO:3) and anucleotide sequence defined by SEQ ID NO:1 or a sequence complementarythereto (SEQ ID NO:4).
 10. A genetically altered plant or progenythereof, wherein the genetically altered plant has reduced alpha, beta,gamma and omega gliadin content compared to the content in acorresponding wild-type plant and comprises the polynucleotide of claim1 or other external genetic material that reduces the expression levelsof genes encoding alpha, beta, gamma and omega gliadins by targeting anucleotide sequence defined by SEQ ID NO:2 or a sequence complementarythereto (SEQ ID NO:3) and a nucleotide sequence defined by SEQ ID NO:1or a sequence complementary thereto (SEQ ID NO:4).
 11. The geneticallyaltered plant of claim 10, wherein the levels of alpha, beta, gamma andomega gliadins in the genetically altered plant are reduced by 20% ormore compared to levels of alpha, beta, gamma and omega gliadins in thecorresponding wild-type plant.
 12. The genetically altered plant ofclaim 10, wherein the genetically altered plant belongs to the genusTriticum.
 13. A seed of the genetically altered plant of claim 10,wherein the seed has reduced alpha-, beta-, gamma-, and omega-gliadincontent.
 14. A pollen, propagule, progeny, or part of the geneticallyaltered plant of claim 10, wherein the pollen, propagule, progeny, orpart of the genetically altered plant has reduced alpha-, beta-, gamma-,and omega-gliadin content.
 15. A food product prepared from the seed ofclaim 13, wherein the food product has reduced alpha-, beta-, gamma-,and omega-gliadin content.
 16. The food product of claim 15, wherein thefood product is flour, a food composition, a vitamin, or a nutritionalsupplement.
 17. A method for preventing the effects of celiac diseasecomprising administering to a patient the food product of claim 16.